EFFECT OF SUBCLINICAL AND CLINICAL MASTITIS ON HAEMATO- BIOCHEMICAL PROFILE AND MILK LEUKOCYTE COUNT IN INDIGENOUS COWS

Journal of Cell and Tissue Research Vol. 16(3) 5829-5834 (2016) (Available online at www. Tcrjournals.com) ISSN: 0973-0028; E-ISSN: 0974-0910 Original Article EFFECT OF SUBCLINICAL AND CLINICAL MASTITIS ON HAEMATO- BIOCHEMICAL PROFILE AND MILK LEUKOCYTE COUNT IN INDIGENOUS COWS SARVESHA, K., 1? SATYANARAYANA, M. L., 1 NARAYANASWAMY, H. D., 1 RAO, S., 1 YATHIRAJ, S., 1 ISLOOR, S., 2 MUKARTAL, S. Y., 2 SRIKANTH, M., 1 ANURADHA, M. E. 2 AND KAMAL, H. 2 1 Department of Veterinary Pathology, 2 Department of Veterinary Microbiology, Veterinary College, Karnataka Veterinary Animal and Fisheries Sciences University (KVAFSU), Bengaluru 560024. E. mail: sarveshkgowda@gmail.com, Cell: 09972201719 Received: September 16, 2016; Accepted: October 7, 2016 Abstract: The study was conducted to determine the effect of subclinical mastitis (SCM) and clinical mastitis (CM) on biochemical parameters and milk leukocyte count of indigenous cows. Milk and blood samples were collected from 20 healthy, 87 SCM and 20 CM affected animals from Kolar and Chikkaballapur districts of Karnataka state, India. The SCM were diagnosed by California Mastitis Test (CMT) and electrical conductivity (EC) of milk. Blood and milk of mastitis infected indigenous cows were analyzed for haemato-biochemical parameters and milk leukocyte count. Haematology revealed significantly (P< 0.05) higher average values of TLC in mastitis infected than healthy animals. Significantly (P< 0.05) lower average values of TEC, Hb and PCV were observed in SCM infected animals, however no significant ((P>0.05) change were observed in values of CM infected than healthy animals. Differential leucocytic count (DLC) revealed higher granulocytes and lymphopenia in mastitis infected animals. Biochemical estimation revealed significantly (P< 0.05) higher average values of Ca, Na and K in mastitis infected compared with healthy animals however, no significant (P> 0.05) change was observed in Mg and ALT levels. AST and TP values were significantly increased in SCM infected compared to healthy animals however, no significant change were observed in CM infected animals. The total milk leukocytes and neutrophils were significantly higher, while lymphocytes and macrophages population were significantly lower in mastitis infected animals. The changes in biochemical parameters and milk leukocyte count are important indicators of the physiological or pathological state (mastitis) of the animal. Key words: Mastitis, Lymphocytes, Cow INTRODUCTION India is one of the leading country in milk production accounting for 18.5 % of global milk production. The crossbred and indigenous cattle contribute 43.7% to the total national milk production. According to the earlier reports low milk production and high environmental adaptability in indigenous cattle contributes to lower prevalence rate of mastitis in comparison to the crossbred cattle and buffaloes but at present due to indiscriminate breeding of indigenous cattle the prevalence rate of mastitis is increasing steadily [1]. Mastitis is the most complex and costly disease of dairy cows occurring throughout the world 5829

J. Cell Tissue Research [2]. Mastitis is an inflammation of the mammary gland of dairy cows accompanied by physical, pathological and bacteriological changes in milk and glandular tissue. In the present state of knowledge it seems practicable and reasonable to define mastitis as a disease characterized by the presence of significantly increased leucocytes content in the milk from affected gland [3]. Mastitis may be clinical or sub-clinical, with subclinical infection preceding clinical manifestation.yet the relationship between the two within a herd is not predictable [4]. Most of the dairy farmers in India are hardly aware of subclinical mastitis and as such attracts no importance to it. In mastitis, there is a break in the blood-milk barrier, along with impaired synthesis and secretary activity of udder epithelial cells, which alters the level of most components. Currently, the information on biochemical profile and milk leukocyte count in indigenous cows affected with mastitis is limited. Therefore, the present study was undertaken. MATERIALS AND METHODS Blood samples were collected from jugular vein of 20 healthy, 87 SCM and 20 CM affected indigenous cows from Kolar and Chikkaballapur districts of Karnataka state, India, in heparinised mineral free (15-20ml) and disodium EDTA (2-3ml) vials. Blood samples were transported to the laboratory within one hour in thermo flask with ice & then fresh blood was analyzed for Hb, PCV, TEC, TLC, ESR and DLC by using fully automated hematology analyzer (Mindray, BC-1800). The serum separated from blood was analyzed for ALT, AST, Ca, Cl, K, Mg, Na, P and TP by using fully automated biochemical analyzer (Thermo scientific KONELAB 20). The milk samples collected were subjected for California Mastitis test (CMT), Somatic cell count (SCC) and electrical conductivity (EC) measurement. EC was determined by using Milk checker (Eisai Co. Ltd. and Oriental instruments Ltd., Tokyo. Japan) and a value more than 6.5 ms/ cm were considered as positive for SCM [5]. The milk samples were collected aseptically in sterilized plastic bottles for SCC and differential leukocyte count. Milk total somatic cell and differential leukocyte count were estimated according to general principle of Prescott and Breed method as detailed by Schalm et al. [6] and a value > 5.00 Lakhs/ml of milk was taken as criteria to declare the milk or animal as subclinically mastitic or infected. Statistical analysis: The data generated from the study were subjected to one way analysis of variance (ANOVA) test using Graph Pad Prism version 5 for windows. Significance of all data was calculated at (P< 0.05). RESULTS Haematology of animals revealed significantly higher average values of TLC in mastitis infected than healthy animals. Significantly lower average values of TEC, Hb and PCV were observed in SCM infected animals, however no significant change were observed in values of CM infected than healthy animals. DLC revealed higher granulocytes and lymphopenia in mastitis infected animals (Table 1). Significantly higher average values of ESR were observed in SCM infected animals, however no significant change were observed in values of CM infected than healthy animals. Biochemical estimation revealed significantly higher average values of Ca, Na, and K in mastitis infected compared with healthy animals however, no significant change was observed in Mg and ALT levels. AST and TP values were significantly increased in SCM infected compared to healthy animals however, no significant change were observed in CM infected animals (Table 2). P and Cl values were significantly decreased in mastitis infected than healthy animals. The total milk leukocytes and neutrophils were significantly higher, while lymphocytes and macrophages population were significantly lower in SCM (Fig. 1) and CM (Fig. 2) infected than healthy animals (Table 3). Significant increase in EC of milk from SCM and CM infected animals were observed in comparison to healthy animals. DISCUSSION Hb, TEC and PCV level of SCM cows showed significant (P< 0.05) decrease in comparison to healthy animals. These findings are in accordance with the findings of Zaki et al. [7] who, reported that anaemia in mastitic cows were due to decrease 5830

Sarvesha et al. Fig. 1: Microscopically subclinical mastitis infected milk showing moderate increase in the total somatic cell count in Modified Newman s stain X 1000. Fig. 2: Microscopically clinical mastitis infected milk showing severe increase in the total somatic cell count in Modified Newman s stain X 1000 in Hb, RBC and PCV levels. No significant changes were observed in Hb and TEC level of CM infected as opposed to healthy animals. The results corroborate with the observation of Sischo et al. [8] who reported that PCV and Hb did not exhibit any specific trend in the animals suffering from mastitis. The ESR of SCM infected animals were found to be higher than healthy animals however, no significant change was observed in CM affected animals. These findings are in agreement with Cebra et al. [9] and Zaki et al. [10] which could be due to persistent infection noticed in SCM. Significant increase in granulocytes and total leukocyte count (TLC) along with decrease in the lymphocyte count were observed in indigenous cows affected with subclinical and clinical mastitis. Increased TLC with increase in absolute number of monocytes, eosinophils and neutrophils in mastitis were reported by various researchers [10,11]. 5831

J. Cell Tissue Research Table 1: Mean ±SE values of haematological profile of control and mastitic indigenous cows. Means marked with different superscript a,b,c differ significantly (P< 0.05) in a row. Sl. No. Parameter Control (n=20) Sub clinical (n=87) Mastitis Clinical (n=20) 1 Hb g/dl 11.36±0.33 a 9.01±0.17 b 10.64±0.34 a 2 PCV % 31.33±0.44 a 28.05±0.25 b 27.40±0.48 b 3 TEC x10 6 /µl 7.99±0.29 a 7.29±0.08 b 7.65±0.15 ab 4 TLC x10 3 /µl 6.83±0.23 a 10.22±0.18 b 11.66±0.31 c 5 Granulocyte % 35.77±0.78 a 65.47±0.28 b 70.53±0.80 c 6 Lymphocyte % 60.50±0.65 a 30.60±0.35 b 25.08±0.89 c 7 Monocyte % 3.74±0.43 3.93±0.21 4.40±0.40 8 ESR mm/2hrs 1.15±0.02 a 1.97±0.02 b 1.20±0.03 a Table 2: Mean ±SE values of biochemical profile of control and mastitic indigenous cows. Means marked with different superscript a,b differ significantly (P< 0.05) in a row. Sl. No. Parameter Control (n=20) Sub clinical (n=87) Mastitis Clinical (n=20) 1 ALT U/L 10.61±0.37 10.50±0.19 10.90±0.31 2 AST U/L 90.73±4.21 a 120.78±1.60 b 91.91±3.04 a 3 TP g/dl 7.23±0.14 a 7.79±0.06 b 7.51±0.17 ab 4 Ca mg/dl 10.50±0.39 a 13.30±0.22 b 13.82±0.22 b 5 P mg/dl 6.89±0.18 a 4.61±0.07 b 4.40±0.13 b 6 Mg mg/dl 2.17±0.10 2.21±0.05 2.16±0.11 7 Cl meq/l 99.09±1.38 a 88.43±0.51 b 89.43±1.15 b 8 Na meq/l 139.51±0.94 a 152.78±0.65 b 153.62±1.07 b 9 K meq/l 4.01±0.10 a 5.66±0.05 b 5.45±0.12 b Table 3: Mean ±SE values of milk EC, SCC and DLC of control and mastitic indigenous cows. Means marked with different superscript a,b,c differ significantly (P< 0.05) in a row. Sl. No. Parameter Control (n=20) Sub clinical (n=87) Mastitis Clinical (n=20) 1 EC ms/cm 4.73±0.21 a 9.95±0.24 b 15.01±0.38 c 2 SCC Lakhs/mL 1.85±0.11 a 8.59±0.43 b 13.14±0.26 c 3 NEUTROPHIL % 4.81±0.24 a 43.59±0.19 b 65.33±0.51 c 4 MACROPHAGE % 76.00±0.14 a 27.88±0.17 b 11.56±0.15 c 5 LYMPHOCYTE % 16.70±0.29 a 17.64±0.18 b 14.29±0.32 c 6 OTHERS % 2.50±0.16 a 10.89±0.32 b 8.83±0.59 c Significant increase in total protein (TP) level of SCM cases compared with healthy animals were observed however, non-significant rise was observed in CM infected animals. Dwivedi et al. [12] reported higher level of TPP (6.81g/dl) and globulin (3.98g/ dl) in serum samples of mastitic cows compared with 6.11g/dl and 3.25g/dl in healthy animals, respectively. Contrary to present findings, a significant decrease in TP was reported by Zaki et al. [10]. This could be due to increased levels of acute phase proteins like haptoglobin, serum amyloid and C reactive proteins, ceruloplasmin, alpha 1-antitrypsin, fibrinogen and decreased albumin following inflammatory response in the body of mastitic cattle [13]. Serum calcium level of the SCM and CM infected animals were significantly (P< 0.05) higher than the healthy animals. This could be due to the decline in the milk yield in infected animals leading to reduced 5832

Sarvesha et al. excretion of calcium in the milk [14]. The findings of this study were in line with Singh et al. [15] who reported significant increased levels of plasma calcium in buffaloes suffering from mastitis. As opposed to present findings Zaki et al. [7] reported significant decrease in the average values of calcium in serum of mastitis infected as compared to healthy cows. Average value of phosphorous of the healthy animals were significantly higher than the SCM and CM infected animals which could be attributed to its higher secretion in milk, due to injury to the udder wall resulting in increased loss in milk which is in accordance with the observation of Dwivedi et al. [12] and Siddiqe et al. [16]. Magnesium level of the serum samples showed no significant effect of mastitis. Dwivedi et al. [12] and Yildiz and Kaygusuzoolu [17] also reported no variation in the plasma level of Mg in mastitic and healthy animals. Compared with present study, Singh [18] reported higher average values of Mg in acute mastitic buffaloes. Contrary to present findings Siddiqe et al. [16] reported significant decrease in the average values of Mg in serum of mastitic cows from that of healthy cows. Sodium and potassium levels of the SCM and CM infected animals were significantly higher than the healthy animals. Atroshi et al. [19] also reported higher serum Sodium and potassium levels in animals suffering from mastitis. This could be due to the decline in the milk yield in animals suffering from mastitis, thereby minimizing the loss of sodium and potassium from body and elevating the level of these ions in the blood. Increased levels of plasma potassium in buffaloes suffering from mastitis was also reported by Singh [18]. Chloride level of the mastitic animals were significantly (P< 0.05) lower than the healthy animals. These findings are in accordance with the findings of Mosallam et al. [20]. This could be due to more loss of Cl in milk due to injury to the udder wall. The highly increases detected in AST values of SCM are in line with the reports of Bayumi et al. [21] which could be due to stressful conditions. The total milk leukocytes and granulocytes were significantly higher, while lymphocytes and macrophages population were significantly lower in mastitis infected animals compared to healthy animals indicating mammary gland infection. The milk leukocyte count is considered the best biomarker for inflammatory reaction in udder infection. The increased number of milk leukocyte in present study could be due to microbial infection in udder as these are the major part of the immune response of the animals [22,23]. These results corroborate with the observation of Hussain et al. [24,25]. The colonization of mammary glands by pathogenic micro-organisms results in a series of events which leads to major alterations of milk compositions secreted from cells. Therefore, CMT is a suitable measure for use on large scale monitoring programs. Significant increase in EC of milk from SCM and CM infected animals were observed compared to healthy animals. This is in accordance with the reports of Sripad et al. [1]. The increase in EC milk of mastitis animals could be due to elevated levels of ions such as sodium, potassium, calcium and chloride during mammary gland inflammation. In conclusion, the changes in haemato-bioche-mical parameters and milk leukocyte count can be used as important indicators of the physiolo-gical or pathological state (mastitis) of the animal. In addition, it is also conclude that CMT, EC and SCC is ideal for early detection of subclinically infected quarters and aids in the selection of dairy animals for either segregation or therapy. Abbreviations used in text: Hemoglobin (Hb), Packed cell volume (PCV), Total erythrocyte count (TEC), Red blood cell (RBC), Total leukocyte count (TLC), Erythrocyte sedimentation rate (ESR), Differential leukocyte count (DLC), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Calcium (Ca), Chloride (Cl), Potassium (K), Magnesium (Mg), Sodium (Na), Phosphorous (P),Total protein(tp), California Mastitis Test (CMT), Electrical conductivity (EC), Somatic cell count (SCC), Subclinical mastitis (SCM) and Clinical mastitis (CM). ACKNOWLEDGEMENTS Authors of the manuscript thankful and acknowledge ICAR NAE for sanctioning project entitled: Animal 5833

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